Detection of enterotoxin A and cytotoxin B, and isolation of Clostridium difficile in piglets in Minas Gerais, Brazil (original) (raw)
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Journal of Swine Health and Production
The aim of this study was to compare test performances of three commercial enzyme immunoassays (EIAs) for A and B toxin detection and that of a simple toxigenic culture protocol to the cytotoxicity assay (CTA) as the gold standard for diagnosis of Clostridium difficile-associated enteritis in piglets. A total of 73 piglets submitted to the Veterinary School of Universidade Federal de Minas Gerais were included in this study. Intestinal content was collected from 62 diarrheic and 11 non-diarrheic piglets, 1 to 7 days old. Vero cells were used in the CTA protocol to detect A and B toxins. Fecal samples were inoculated on cycloserine-cefoxitin fructose agar for isolation of C difficile. The EIAs were performed according to the manufacturers' instructions. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated for each EIA and for toxigenic culture against CTA.
Clostridium difficile is a Gram-positive opportunistic anaerobic bacterium found in the soil, water as well as the digestive tract of several species of mammals. Currently in high-health herds and not related with antibiotic treatment, C. difficile has been emerged in association with neonatal catarrhal, fibrinous or purulent colitis in 2 to 7 day-old piglets. Mesocolon edema and colitis with a " volcano" lesion are pathological key marks of C. difficile infection. In farm 1, the prevalence of neonatal piglet mesocolon edema and colitis was evaluated in an extensive study of postmortem preweaning mortality. Lectinhistochemistry pattern of normal colon and colon with edema and colitis were analyzed in order to provide more accurate information related with pathogenesis of C.difficile infection. In farm 2, a clinical description of an outbreak of neonatal colitis in piglets due to C. difficile infection was reported. A total 820 piglets were post-mortem examined, from them, 8 cases were classified as suspected of C. difficile infection (0.1%). Age of affected piglets varied between 3 to 14 days. In all of them, the key mark was the severe mesocolon edema and 2 cases were characterized by focal necrosis and loss of epithelial cells associated with a focal infiltration of neutrophils and macrophages in the lamina propria and lumen like erupted volcano. Lectins SBA and DBA that has affinity to α Gal epitope were negative in normal samples but reacted strongly positive at the glycocalix of the villi enterocytes of the affected samples indicating a change in the carbohydrates pattern of the cells surfaces that might favor the binding of C. difficile toxin A (TcdA). In farm 2, from 11 examined piglets, 6 of them (54%) showed different degree of mesocolon edema and in only 2 of them; volcano lesions were the main histopathological findings. Rectal swab from 8 diarrheic and 5 postmortem examined piglets were surveyed for eltA estI estII and stx 2e virulence gens of E. coli and all samples were negative. Clostridium difficile toxins A and B were identified only in the sample with severe gross and microscopic changes. Isolation of C. difficile was unsuccessfully. Infection of C. difficile is present in Argentinean pig farms. Diagnosis of subclinical infection might be negligent if post-mortem studies of preweaning mortality at weekly intervals are not performed. However, when clinical disease appeared, mortality might be high as it was reported in the farm 2. Comparative lectinhistochemistry studies from field cases added further information of the carbohydrates present on glycocalix of villi enterocytes related with toxin receptors.
Current Science, 2017
Clostridium difficile with its virulence factors A and B toxins cause Pseudomembranous colitis. Bacterium was isolated from 57 dog and 41 pig diarrheic faecal samples in cycloserinecefaxitin fructose agar media and molecular detection was done by amplification of gluD gene (755 bp). Variability of toxin genes in positive isolates was tested by multiplex PCR. Detection of binary toxin genes (cdtA and cdtB) was also done. Results showed 33.67% positivity with 18 and 15 from dog and pig respectively, from which 10 and 5 were toxigenic and 11 pig isolates exhibited binary toxin. PCR-RFLP demonstrated toxinotype 0 in all A + B + isolates.
Clostridium difficile with its virulence factors A and B toxins cause Pseudomembranous colitis. Bacterium was isolated from 57 dog and 41 pig diarrheic faecal samples in cycloserinecefaxitin fructose agar media and molecular detection was done by amplification of gluD gene (755 bp). Variability of toxin genes in positive isolates was tested by multiplex PCR. Detection of binary toxin genes (cdtA and cdtB) was also done. Results showed 33.67% positivity with 18 and 15 from dog and pig respectively, from which 10 and 5 were toxigenic and 11 pig isolates exhibited binary toxin. PCR-RFLP demonstrated toxinotype 0 in all A + B + isolates.
International Journal of Food Microbiology, 2013
The objective of this study was to evaluate the presence of Clostridium difficile in intestinal and carcass samples collected from pigs and cattle at a single slaughterhouse. C. difficile was isolated in 1% and 9.9% of the pig and cattle intestinal contents and in 7.9% and 7% of cattle and pig carcass samples respectively. A total of 19 different PCR-ribotypes were identified, among them types 078 and 014. Seven of 19 ribotypes correlated with the PCRribotypes involved in human C. difficile infections in Belgium. This study confirms that animals are carriers of C. difficile at slaughter and ribotypes are identical than those in humans, and that carcass contamination occurs inside the slaughterhouse.
PCR-ribotype distribution of Clostridium difficile in Irish pigs
Anaerobe, 2017
Clostridium difficile is an important enteric pathogen in humans causing infections in the healthcare environment and the community. Carriage of C. difficile and C. difficile-related enterocolitis has been reported in piglets worldwide. The aim of this study was to investigate the rates of C. difficile isolation from pigs in Ireland. Faecal samples from piglet litters and sows were collected from six farms in 2015. The sows were non-diarrhoeal at the time of sampling. The diarrhoeal status of the piglets was unknown. C. difficile was isolated from 34/44 (77%) of piglet litter samples and from 33/156 (21%) of sow samples. The isolation rate in sows varied from 3 to 39% and in piglet litters from 72 to 86% depending on farm location. Toxin A and toxin B were present in 99% (66/67) of isolates; and binary toxin in 85% (57/67). Only PCR-ribotypes 078 (88%) and 193 (12%) were identified in piglets. Seven PCR-ribotypes were detected in sow C. difficile isolates: PCR-ribotypes 078 (67%), 0...
Advances in Microbiology, 2013
Clostridium difficile and C. perfringens are enteric pathogens affecting a variety of mammals. This study evaluated the molecular enterotoxigenicity of Clostridium swine isolates by PCRs. One hundred and ten swine faeces were analyzed by culture assay. The faecal samples were from sixty-seven healthy animals and 43 with gastrointestinal tract disease. C. difficile strains were PCR-screened for the presence of tcdA/tcdB and cdtA/cdtB genes. All C. perfringens isolates were tested for the characterization of the toxinotype. Overall, sixty-five swine resulted positive: 38 for C. difficile and 17 for C. perfringens. One sample tested C. perfringens and C. difficile-positive, at the same time: on the whole, 39 C. difficile strains were isolated. Thirty-eight C. difficile isolates (all from healthy animals) resulted tcdA/tcdB and cdtA/cdtBnegative by PCRs and toxins A/B-negative by immunological tests. All C. perfringens strains were type A; eight were also cpb2-positive. In the sample (diarrhoeic), with double infection, C. difficile tested tcdA/tcdB and cdtA/cdtB-positive by PCRs and toxins A/B-positive by immunoassays; C. perfringens resulted cpb2-positive. The molecular genotypeing/toxinotyping should be applied to establish a final diagnosis and to assess properly the full implications and the epidemiological impact of these findings in particular in samples of healthy animals and aid in the development of effective intervention methods for controlling clostridial disease outbreaks.
Clostridium difficile in Swine: Zoonotic Transfer and the Potential Consequences
2019
Clostridium difficile is an anaerobic, gram-positive, spore-forming bacterium that can infect both animals and humans. C. difficile infection (CDI) is a toxin-mediated disease that can result in the production of three main virulence factors: toxin A, toxin B and binary toxin. Newborn piglets are highly susceptible to CDI, however age decreases the chances of colonization. Prevention and treatment strategies are limited, and there is currently no commercially available treatment option for CDI. However, treatment methods and prevention strategies using a nontoxigenic C. difficile strain and equine-origin antitoxins have been explored and show preliminary promising results. With evidence of possible zoonotic transfer increasing, agriculture and medical professionals should take action to prevent the spread of C. difficile. Consequences including economic loss and decline in consumer confidence could result if a highly virulent resistant strain of C. difficile emerged and caused an increase in morbidity and mortality rates among pigs and humans. Clostridium difficile in Neonatal Piglets Clostridium difficile is an anaerobic, gram-positive, spore-forming bacterium present in both animals and humans (Squire and Riley 2013). Clostridium difficile infection (CDI) occurs in organisms with a compromised immune system and those with disrupted gut floras. C. difficile is one of the leading causes of enteritis in newborn piglets and it produces three main virulent toxins: toxin A, toxin B, and binary toxin (Abt, McKenney and Pamer 2016). Colonization can occur within 48 hours of birth in virtually 100% of neonates (Hopman et al. 2011). Within swine herds, C. difficile is shown to have the ability to impact, on average, two-thirds of litters, causing
Advances in Microbiology
Clostridium difficile and C. perfringens are enteric pathogens affecting a variety of mammals. This study evaluated the molecular enterotoxigenicity of Clostridium swine isolates by PCRs. One hundred and ten swine faeces were analyzed by culture assay. The faecal samples were from sixty-seven healthy animals and 43 with gastrointestinal tract disease. C. difficile strains were PCR-screened for the presence of tcdA/tcdB and cdtA/cdtB genes. All C. perfringens isolates were tested for the characterization of the toxinotype. Overall, sixty-five swine resulted positive: 38 for C. difficile and 17 for C. perfringens. One sample tested C. perfringens and C. difficile-positive, at the same time: on the whole, 39 C. difficile strains were isolated. Thirty-eight C. difficile isolates (all from healthy animals) resulted tcdA/tcdB and cdtA/cdtBnegative by PCRs and toxins A/B-negative by immunological tests. All C. perfringens strains were type A; eight were also cpb2-positive. In the sample (diarrhoeic), with double infection, C. difficile tested tcdA/tcdB and cdtA/cdtB-positive by PCRs and toxins A/B-positive by immunoassays; C. perfringens resulted cpb2-positive. The molecular genotypeing/toxinotyping should be applied to establish a final diagnosis and to assess properly the full implications and the epidemiological impact of these findings in particular in samples of healthy animals and aid in the development of effective intervention methods for controlling clostridial disease outbreaks.